Matt:LabNotes/2015-2-5: Difference between revisions
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>Mzcai |
>Mzcai m (→DpnII) |
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Line 105: | Line 105: | ||
*Added 5ul DpnII | *Added 5ul DpnII | ||
*Incubated at 37C for overnight (~15hrs) | *Incubated at 37C for overnight (~15hrs) | ||
===Zymo Column Purification=== | ===Zymo Column Purification=== | ||
*Eluted 30ul each column (115ul total) | *Eluted 30ul each column (115ul total) | ||
*Nanodrop | *Nanodrop | ||
** | **ng/ul x 115ul = ug (% yield) | ||
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===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run 3 gels | *Run 3 gels |
Revision as of 19:04, 9 February 2015
CA12k_Nov2014_V7 Probe Production
- Production of Padlock Probes (V7) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
- Do double of what I normally do (96 100ul PCR reactions instead of 48)
Production PCR
- 10nM 1st round amplicons
- V7 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V7 (10nM) | 0.2 | 20 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V7U (100uM) | 0.4 | 40 |
AP2V7 (100uM) | 0.4 | 40 |
H2O | 49 | 4900 |
Total | 100 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]
EtOH Precipitation
- 12 5-ml tubes (with 8 wells of PCR product each) for V7
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 90min
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~590ul of V7 probes: 107.3ng/ul => 63.3ug
Lambda Exonuclease Digestion
- Divide into 6 pcr tubes of ~100ul with total amplicon of ~10ug each
Components | Volume |
Amplicon | 98 |
10X Lambda Exo Buffer | 12 |
Lambda Exonuclease | 12 |
Total | 122 |
- Incubated at 37C for 1hr
- Purified with 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 78.1ng/ul x 240ul = 18.7ug (% yield)
Remove Amplification Adapters
USER
- Split into 4 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 59 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 8 |
Total | 80 |
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 2 |
100uM RE-DpnII_V7 guide oligo | 5 |
H2O | 8 |
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (~15hrs)
Zymo Column Purification
- Eluted 30ul each column (115ul total)
- Nanodrop
- ng/ul x 115ul = ug (% yield)